Accompanying recent remarkable progress in molecular biology, various mechanisms of diseases are being clarified on molecular level, and chemotherapy of cancer has entered upon a new stage. That is, therapy method specified for each individual patient, which is referred to as tailor-made therapy, is in demand, and to meet that demand, it is essential to establish drug design aiming at alleviation of side effect based on clarification of molecular theoretical mechanisms or methodology for drug administration. Generally chemotherapy using carcinostatic or anticancer agents is known as a therapeutic method indispensable where radiotherapy achieves only insufficient effect or where operation cannot be applied as in the case of leukemia.
However, under the current status, cancer chemotherapy is subject to many problems compared to the success in antibiotic therapy represented by penicillin and streptomycin. The greatest reason is that the most of anticancer drugs possess potent toxicity to human body and hence attempts to improve the therapeutic effect by increasing administration doses accompany undesirable side effect, which sets a limit to pharmacotherapy. Under the circumstances, an important point is how to reduce toxicity of anticancer drugs themselves and to promote the therapeutic effect. It is the recent tendency in chemotherapy of cancer developed from the foregoing background that drug targeting aiming at improvement in selectivity of anticancer drugs for target cancer cells is gathering attention.
Folic acid is a member of vitamin B group known as having various physiological activities, which is transported into the cells via the mechanisms called endocytosis or potocytosis mediated by folic acid receptors which are present at the cell surfaces. Hence, if a drug can be bonded with folic acid (formation of folic acid-drug conjugate), positive transport of the drug into the cells via these mechanisms would become possible. Furthermore, it is known that the receptors which recognize folic acid are excessively expressed in cancer cells, and folic acid-drug conjugates are expected to be capable of targeting cancer cells. For example, systems in which folic acid is bound to doxorubicin (DOX, trivial name: adriamycin) or antisense oligodeoxynucleotide (ODN) have already been under investigation and their drug targeting effects are demonstrated (e.g., see the later-identified non-patent documents 1 and 2).
Thus, biological approach using folic acid involves many aspects of high interest, and importance of such folic acid derivatives is widely recognized. Whereas, differing from those conjugates disclosed in the non-patent documents 1 and 2, a number of problems have been pointed out in respect of syntheses of such conjugates heretofore obtained by direct covalent bonding of folic acid with drug. Namely, in most cases covalent bonding of folic acid with drug is conducted with use of a condensing agent such as DCC, and the products are frequently obtained as α- and γ-carboxylate mixtures, and it is very difficult to purify the aimed compound alone as contained in the mixtures. Furthermore, alpha-folic acid derivatives are considered to be entirely meaningless for application in biochemical field, because they have no ability to recognize receptors. While a number of synthesis methods of folic acid derivatives containing γ-carboxylate alone were reported, they generally involve long reaction steps and lack versatility.
Of these, production methods promising to a certain extent also are proposed. For example, in one of them pteroylazide, which was formed from pteroic acid corresponding to the pteroyl moiety as a part of folic acid structure gave folate γ-methyl ester as a key intermediate, through the reaction with γ-methyl glutamate. The intermediate thus obtained was subsequently reacted with ethylenediamine, followed by the conjugation with tumor-specific metal binding ligand (DTPA) via the free amino groups at the ethylenediamine terminal. The literature disclosing the above method also disclosed the compounds obtained by the same method (see, for example, later-identified non-patent document 3).
However, these production methods require many reaction stages before obtaining the key intermediate, and the key intermediate itself is almost insoluble in organic solvents customarily used in organic synthesis reactions. On the other hand, Nomura, M. et al. first pointed out not only the low solubility of the key intermediate in an organic solvent but also the relatively low reactivity between γ-methyl ester moiety with nucleophilic agent, and then proposed a method for obtaining an intermediate product corresponding to the key intermediate, which comprises first protecting the 2-amino group of pteridine ring in pteroic acid with oleophilic group, then converting its carboxyl group to imidazolide, and reacting the imidazolide with a glutamic acid derivative in which γ-carboxyl group of glutamic acid is retained in free state and α-carboxyl group is protected with oleophilic group (see, for example, later-identified non-patent document 4).
Nomura, et al. obtained with use of a condensing agent, a conjugate whose covalent bond is formed between free γ-carboxyl group of such an intermediate and amino group of a drug. However, multi-stage steps are required for obtaining the glutamic acid derivative in which γ-carboxyl group is maintained in free state and α-carboxyl group is protected with an oleophilic group.
List of Cited Documents
    Non-patent document 1: Lee, R. J. et al., J. Boil. Chem. 1994, 269, 3198-3204    Non-patent document 2: Wang, S. et al., Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 3318-3322    Non-patent document 3: Luo, J. et al., J. Am. Chem. Soc. 1997, 119, 10004-10013    Non-patent document 4: Nomura, M. et. al., J. Org. Chem. 2000, 65, 5016-5021